Determination of Virus Abundance in Marine Sediments

In this study, we optimized procedures to enumerate viruses from marine sediments by epifluorescence microscopy using SYBR Green I as a stain. The highest virus yields from the bulk of the sediments were obtained by utilizing pyrophosphate and 3 min of sonication. The efficiency of extraction benthic viruses by pyrophosphate-ultrasound treatment was about 60% of the extractable virus particles. Samples treated with nucleases had increased virus counts, suggesting a masking effect of extracellular DNA. No significant differences were observed between virus counts obtained by epifluorescence microscopy and transmission electron microscopy. Both formaldehyde and glutaraldehyde gave significant reductions of virus counts after only 24 h of sediment storage, but no further loss occurred after 7 days.     

Flow cytometric analysis of bacteria- and virus-like particles in lake sediments

Flow cytometry (FCM) was successfully used to analyze freshwater bacteria and viruses in lake sediments after relatively simple sample treatment and optimization of dilution/fixation/staining procedures. Biological particles from Lakes Geneva and Bourget were first separated from the sediments by using both Sodium Pyrophosphate (0.01 M final concentration) and Polyoxyethylene-Sorbitan Monooleate (10% final concentration) and sonicating for 3 min in a water bath. The best results (based on FCM signature and the highest virus and bacterial yields from the sediments) were obtained by formaldehyde fixation carried out within less than one hour (2% final concentration, vs. no fixation or using glutaraldehyde at different concentrations), SYBR-Green II staining (x1/20,000 stock solution concentration, vs. use of SYBR-Gold and SYBR-Green I dyes at different concentrations). There was a considerable loss of particles after only a few days of storage at either 4 or -22 degrees C. For FCM analysis, the samples were diluted in Tris-EDTA buffer (pH 8) and heated for 10 min at 75 degrees C after incubating for 5 min in the dark. The bacterial and viral counts paralleled those obtained using epifluorescence microscopy (EFM), but EFM always gave lower counts than FCM. Analysis of the distribution of the viruses in the water column and in the sediments of Lakes Bourget revealed a marked gradient, with larger quantities in the top layer of the sediment than in the water above it. These results are discussed, as well as the possible novel application of flow cytometry in the study of aquatic viral ecology.     

Enumeration of Marine Viruses in Culture and Natural Samples by Flow Cytometry

Flow cytometry (FCM) was successfully used to enumerate viruses in seawater after staining with the nucleic acid-specific dye SYBR Green-I. The technique was first optimized by using the Phaeocystis lytic virus PpV-01. Then it was used to analyze natural samples from different oceanic locations. Virus samples were fixed with 0.5% glutaraldehyde and deep frozen for delayed analysis. The samples were then diluted in Tris-EDTA buffer and analyzed in the presence of SYBR Green-I. A duplicate sample was heated at 80°C in the presence of detergent before analysis. Virus counts obtained by FCM were highly correlated to, although slightly higher than, those obtained by epifluorescence microscopy or by transmission electron microscopy (r = 0.937, n = 14, and r = 0.96, n = 8, respectively). Analysis of a depth profile from the Mediterranean Sea revealed that the abundance of viruses displayed the same vertical trend as that of planktonic cells. FCM permits us to distinguish between at least two and sometimes three virus populations in natural samples. Because of its speed and accuracy, FCM should prove very useful for studies of virus infection in cultures and should allow us to better understand the structure and dynamics of virus populations in natural waters.     

Abundance of Viruses in Marine Waters: Assessment by Epifluorescence and Transmission Electron Microscopy

Abundance of bacteria and tiny DNA-associated particles in the upper layer of Japanese coastal and offshore waters was evaluated by epifluorescence microscopy with 0.015-μm-pore-size Nuclepore filters. The number of tiny DNA-associated particles was compared with the abundance of virus particles estimated by transmission electron microscopy. Although a large variation in virus abundance (1.2 × 10⁶ to 35 × 10⁶ ml⁻¹) was obtained with the transmission electron microscopy method, the ratio of 4′,6-diamidino-2-phenylindole-reactive tiny particles to viruses was in a rather narrow range (1.0 to 1.6), indicating that the majority of the tiny DNA-associated particles identified by epifluorescence microscopy were actually virus particles. This result implies the possibility of using epifluorescence microscopy for the evaluation of virus abundance in marine environments.     

Barophilic Bacteria Associated with Digestive Tracts of Abyssal Holothurians

Abyssal holothurians and sediment samples were collected at depths of 4,430 to 4,850 m in the Demerara abyssal plain. Bacterial concentrations in progressive sections of the holothurian digestive tract, as well as in surrounding surface sediments, were determined by epifluorescence microscopy. Total bacterial counts in sediments recently ingested by the animals were 1.5- to 3-fold higher than in surrounding sediments at the deepest station. Lowest counts were observed consistently in the foregut, where the digestive processes of the holothurian are believed to occur. In most animals, counts increased 3- to 10-fold in the hindgut. Microbial activity at 3°C and in situ and atmospheric pressure were determined for gut and sediment samples by measuring the utilization of [¹⁴C]glutamic acid, the doubling time of the mixed-population of culturable bacteria, and the percentage of the total bacterial count responsive to yeast extract in the presence of nalidixic acid, using epifluorescence microscopy. A barophilic microbial population, showing elevated activity under deep-sea pressure, was detected by all three methods in sediments removed from the hindgut. Transmission electron micrographs revealed intact bacteria directly associated with the intestinal lining only in the hindgut. The bacteria are believed to be carried as an actively metabolizing, commensal gut flora that transforms organic matter present in abyssal sediments ingested by the holothurian. Using data obtained in this study, it was calculated that sediment containing organic matter altered by microbial activity cleared the holothurian gut every 16 h, suggesting that abyssal holothurians and their associated gut flora are important participants in nutrient cycles of the abyssal benthic ocean.     

Distribution of virus-like Particles in an Oligotrophic Marine Environment (Alboran Sea,Western Mediterranean)

Viruses are abundant in a variety of aquatic environments, often exceeding bacterial abundance by one order of magnitude. In the present study, the spatial distribution of viruses in offshore waters of the Alboran Sea (Western Mediterranean) have been studied to determine the relationships between viruses and host communities in this oligotrophic marine environment. Viral abundance was determined using two methods: (i) epifluorescence light microscopy using the dsDNA binding fluorochrome DAPI, and (ii) direct counts by transmission electron microscopy (TEM). The results obtained were significantly different; the highest viral counts were obtained by mean of TEM analyses. In all the samples tested the number of viruses was exceeded by the bacterial concentrations, with a ratio between viral and bacterial titers varying between 1.4 and 20. VLP (virus-like particle) counts were not significantly correlated (p > 0.001) with chlorophyll a concentration or the abundance of cyanobacteria. However, there was a positive and significant correlation with bacterial abundance (p < 0.001). The analysis of size and morphology of viral particles by TEM and the correlation obtained between the numbers of VLP and bacteria suggest that the majority of the viral particles in the Alboran Sea are bacteriophages. None of the indirect evidence suggested that eukaryotic algae or cyanobacteria were important host organisms in these waters.     

Application of digital image analysis and flow cytometry to enumerate marine viruses stained with SYBR gold

A novel nucleic acid stain, SYBR Gold, was used to stain marine viral particles in various types of samples. Viral particles stained with SYBR Gold yielded bright and stable fluorescent signals that could be detected by a cooled charge-coupled device camera or by flow cytometry. The fluorescent signal strength of SYBR Gold-stained viruses was about twice that of SYBR Green I-stained viruses. Digital images of SYBR Gold-stained viral particles were processed to enumerate the concentration of viral particles by using digital image analysis software. Estimates of viral concentration based on digitized images were 1.3 times higher than those based on direct counting by epifluorescence microscopy. Direct epifluorescence counts of SYBR Gold-stained viral particles were in turn about 1.34 times higher than those estimated by the transmission electron microscope method. Bacteriophage lysates stained with SYBR Gold formed a distinct population in flow cytometric signatures. Flow cytometric analysis revealed at least four viral subpopulations for a Lake Erie sample and two subpopulations for a Georgia coastal sample. Flow cytometry-based viral counts for various types of samples averaged 1.1 times higher than direct epifluorescence microscopic counts. The potential application of digital image analysis and flow cytometry for rapid and accurate measurement of viral abundance in aquatic environments is discussed.     

Application of Digital Image Analysis and Flow Cytometry To Enumerate Marine Viruses Stained with SYBR Gold

A novel nucleic acid stain, SYBR Gold, was used to stain marine viral particles in various types of samples. Viral particles stained with SYBR Gold yielded bright and stable fluorescent signals that could be detected by a cooled charge-coupled device camera or by flow cytometry. The fluorescent signal strength of SYBR Gold-stained viruses was about twice that of SYBR Green I-stained viruses. Digital images of SYBR Gold-stained viral particles were processed to enumerate the concentration of viral particles by using digital image analysis software. Estimates of viral concentration based on digitized images were 1.3 times higher than those based on direct counting by epifluorescence microscopy. Direct epifluorescence counts of SYBR Gold-stained viral particles were in turn about 1.34 times higher than those estimated by the transmission electron microscope method. Bacteriophage lysates stained with SYBR Gold formed a distinct population in flow cytometric signatures. Flow cytometric analysis revealed at least four viral subpopulations for a Lake Erie sample and two subpopulations for a Georgia coastal sample. Flow cytometry-based viral counts for various types of samples averaged 1.1 times higher than direct epifluorescence microscopic counts. The potential application of digital image analysis and flow cytometry for rapid and accurate measurement of viral abundance in aquatic environments is discussed.     

High virus-to-cell ratios indicate ongoing production of viruses in deep subsurface sediments

Marine sediments cover two-thirds of our planet and harbor huge numbers of living prokaryotes. Long-term survival of indigenous microorganisms within the deep subsurface is still enigmatic, as sources of organic carbon are vanishingly small. To better understand controlling factors of microbial life, we have analyzed viral abundance within a comprehensive set of globally distributed subsurface sediments. Phages were detected by electron microscopy in deep (320 m below seafloor), ancient (∼14 Ma old) and the most oligotrophic subsurface sediments of the world''s oceans (South Pacific Gyre (SPG)). The numbers of viruses (10⁴–10⁹ cm⁻³, counted by epifluorescence microscopy) generally decreased with sediment depth, but always exceeded the total cell counts. The enormous numbers of viruses indicate their impact as a controlling factor for prokaryotic mortality in the marine deep biosphere. The virus-to-cell ratios increased in deeper and more oligotrophic layers, exhibiting values of up to 225 in the deep subsurface of the SPG. High numbers of phages might be due to absorption onto the sediment matrix and a diminished degradation by exoenzymes. However, even in the oldest sediments, microbial communities are capable of maintaining viral populations, indicating an ongoing viral production and thus, viruses provide an independent indicator for microbial life in the marine deep biosphere.     

Distribution of viruses in the Chesapeake Bay.

High virus counts were found in water samples collected from the Chesapeake Bay. Viruses were enumerated by ultracentrifugation of water samples onto grids which were visualized by transmission electron microscopy. Virus counts in September 1990, April 1991, June 1991, August 1991, and October 1991 ranged between 2.6 x 10(6) and 1.4 x 10(8) viruses ml-1 with a mean of 2.5 x 10(7) viruses ml-1. Virus counts were usually at least three times higher than direct bacterial counts in corresponding samples. Virus counts in August and October were significantly higher than at the other sampling times, whereas bacterial counts were significantly lower at that time, yielding mean virus-to-bacterium ratios of 12.6 and 25.6, respectively. From analysis of morphology of the virus particles, it is concluded that a large proportion of the viruses are bacteriophages. The high virus counts obtained in this study suggest that viruses may be an important factor affecting bacterial populations in the Chesapeake Bay, with implications for gene transfer in natural aquatic bacterial populations and release of genetically engineered microorganisms to estuarine and coastal environments.     

Distribution of viruses in the Chesapeake Bay.

High virus counts were found in water samples collected from the Chesapeake Bay. Viruses were enumerated by ultracentrifugation of water samples onto grids which were visualized by transmission electron microscopy. Virus counts in September 1990, April 1991, June 1991, August 1991, and October 1991 ranged between 2.6 x 10(6) and 1.4 x 10(8) viruses ml-1 with a mean of 2.5 x 10(7) viruses ml-1. Virus counts were usually at least three times higher than direct bacterial counts in corresponding samples. Virus counts in August and October were significantly higher than at the other sampling times, whereas bacterial counts were significantly lower at that time, yielding mean virus-to-bacterium ratios of 12.6 and 25.6, respectively. From analysis of morphology of the virus particles, it is concluded that a large proportion of the viruses are bacteriophages. The high virus counts obtained in this study suggest that viruses may be an important factor affecting bacterial populations in the Chesapeake Bay, with implications for gene transfer in natural aquatic bacterial populations and release of genetically engineered microorganisms to estuarine and coastal environments.     

Assessment of Factors Influencing Direct Enumeration of Viruses within Estuarine Sediments

Accurate enumeration of viruses within environmental samples is critical for investigations of the ecological role of viruses and viral infection within microbial communities. This report evaluates differences in viral and bacterial direct counts between estuarine sediment samples which were either immediately processed onboard ship or frozen at −20°C and later processed. Viral and bacterial abundances were recorded at three stations spanning the length of the Chesapeake Bay in April and June 2003 within three sediment fractions: pore water (PW), whole sediment (WS), and sediment after pore water removal (AP). No significant difference in viral abundance was apparent between extracts from fresh or frozen sediments. In contrast, bacterial abundance was significantly lower in the samples subjected to freezing. Both bacterial and viral abundance showed significant differences between sediment fractions (PW, WS, or AP) regardless of the fresh or frozen status. Although pore water viral abundance has been used in the past as a measurement of viral abundance in sediments, this fraction accounted for only ca. 5% of the total sediment viral abundance across all samples. The effect of refrigerated storage of sediment viral extracts was also examined and showed that, within the first 2 h, viral abundance decreased ca. 30% in formalin-fixed extracts and 66% in unfixed extracts. Finally, the reliability of direct viral enumeration via epifluorescence microscopy was tested by using DNase treatment of WS extractions. These tests indicated that a large fraction (>86%) of the small SYBR gold fluorescing particles are likely viruses.     

Assessment of factors influencing direct enumeration of viruses within estuarine sediments

Accurate enumeration of viruses within environmental samples is critical for investigations of the ecological role of viruses and viral infection within microbial communities. This report evaluates differences in viral and bacterial direct counts between estuarine sediment samples which were either immediately processed onboard ship or frozen at -20 degrees C and later processed. Viral and bacterial abundances were recorded at three stations spanning the length of the Chesapeake Bay in April and June 2003 within three sediment fractions: pore water (PW), whole sediment (WS), and sediment after pore water removal (AP). No significant difference in viral abundance was apparent between extracts from fresh or frozen sediments. In contrast, bacterial abundance was significantly lower in the samples subjected to freezing. Both bacterial and viral abundance showed significant differences between sediment fractions (PW, WS, or AP) regardless of the fresh or frozen status. Although pore water viral abundance has been used in the past as a measurement of viral abundance in sediments, this fraction accounted for only ca. 5% of the total sediment viral abundance across all samples. The effect of refrigerated storage of sediment viral extracts was also examined and showed that, within the first 2 h, viral abundance decreased ca. 30% in formalin-fixed extracts and 66% in unfixed extracts. Finally, the reliability of direct viral enumeration via epifluorescence microscopy was tested by using DNase treatment of WS extractions. These tests indicated that a large fraction (>86%) of the small SYBR gold fluorescing particles are likely viruses.     

An Inexpensive,Accurate, and Precise Wet-Mount Method for Enumerating Aquatic Viruses

Viruses affect biogeochemical cycling, microbial mortality, gene flow, and metabolic functions in diverse environments through infection and lysis of microorganisms. Fundamental to quantitatively investigating these roles is the determination of viral abundance in both field and laboratory samples. One current, widely used method to accomplish this with aquatic samples is the “filter mount” method, in which samples are filtered onto costly 0.02-μm-pore-size ceramic filters for enumeration of viruses by epifluorescence microscopy. Here we describe a cost-effective (ca. 500-fold-lower materials cost) alternative virus enumeration method in which fluorescently stained samples are wet mounted directly onto slides, after optional chemical flocculation of viruses in samples with viral concentrations of <5 × 10⁷ viruses ml⁻¹. The concentration of viruses in the sample is then determined from the ratio of viruses to a known concentration of added microsphere beads via epifluorescence microscopy. Virus concentrations obtained by using this wet-mount method, with and without chemical flocculation, were significantly correlated with, and had precision equivalent to, those obtained by the filter mount method across concentrations ranging from 2.17 × 10⁶ to 1.37 × 10⁸ viruses ml⁻¹ when tested by using cultivated viral isolates and natural samples from marine and freshwater environments. In summary, the wet-mount method is significantly less expensive than the filter mount method and is appropriate for rapid, precise, and accurate enumeration of aquatic viruses over a wide range of viral concentrations (≥1 × 10⁶ viruses ml⁻¹) encountered in field and laboratory samples.     

Evaluation of membrane adsorption-epifluorescence microscopy for the enumeration of bacteria in coastal surface films

We have evaluated a method for enumerating surface slick bacteria by combining a membrane adsorption procedure with epifluorescence microscopy. Various chemicals were investigated for their ability to enhance bacterial elution from the membrane filters. The results of the elution-epifluorescence method were compared to plate counts and to direct epifluorescence counts of the sampling membrane filters. In all tests, the elution-epifluorescence technique yielded significantly higher bacterial concentrations.     

Virus and prokaryote enumeration from planktonic aquatic environments by epifluorescence microscopy with SYBR Green I

Viruses are the most abundant biological entities in aquatic environments, typically exceeding the abundance of bacteria by an order of magnitude. The reliable enumeration of virus-like particles in marine microbiological investigations is a key measurement parameter. Although the size of typical marine viruses (20-200 nm) is too small to permit the resolution of details by light microscopy, such viruses can be visualized by epifluorescence microscopy if stained brightly. This can be achieved using the sensitive DNA dye SYBR Green I (Molecular Probes-Invitrogen). The method relies on simple vacuum filtration to capture viruses on a 0.02-microm aluminum oxide filter, and subsequent staining and mounting to prepare slides. Virus-like particles are brightly stained and easily observed for enumeration, and prokaryotic cells can easily be counted on the same slides. The protocol provides an inexpensive, rapid (30 min) and reliable technique for obtaining counts of viruses and prokaryotes simultaneously.     

Proportion of prokaryotes enumerated as viruses by epifluorescence microscopy

It is well known that there are prokaryotes small in size (e.g. ultra-microprokaryotes) that pass through a 0.2-μm filter. As bacterial and viral abundances are determined by epifluorescence microscopy and the differentiation between them is based on particle size, some bacteria can be erroneously enumerated as viruses, namely in marine waters where bacteria are small. However, there is no information on the proportion of prokaryotes that could be misidentified as viruses by epifluorescence microscopy. In this work, we assessed, in water samples collected in the estuarine system Ria de Aveiro (Portugal), the proportion of prokaryotes that could be counted as viruses by the current widespread epifluorescence microscopy and, for the first time, by fluorescence in situ hybridization (FISH). The total number of particles was determined on membranes of 0.2 and 0.02 μm after staining with 4′,6-diamidino-2-phenylindole (DAPI), and the number of prokaryotes (Bacteria and Archaea) was determined by FISH for both pore size membranes. The results show that, in the marine zone of the estuarine system, 28 % of particles enumerated as virus-like particles were prokaryotes, but, in the brackish water zone, only 13 % of the particles counted as viruses were actually prokaryotic cells. Epifluorescence microscopy overestimates viral abundance, and also the ratio viruses:prokaryotes, and this error must be taken into consideration because it can vary significantly within a system. In fact, in the marine zone of an estuarine system, the overestimation of viral abundance can be twice as high as in the brackish water zone.     

Microbial community structure and ecology of subglacial sediments in two polythermal Svalbard glaciers characterized by epifluorescence microscopy and PLFA

Biological and physico-chemical characteristics of subglacial sediments were studied in Svalbard. Sediment from close proglacial and supraglacial environments was used for a comparison. Viable bacteria, cyanobacteria and microalgae were detected in subglacial sediments from two polythermal glaciers using epifluorescence microscopy and phospholipid fatty acid (PLFA) analyses. The subglacial samples were generally of higher pH values, coarser texture and lower water content, organic matter, organic carbon, and nitrogen compared to proglacial and supraglacial sediments). Bacterial counts of 1.6 × 10⁷ cells mg^{− 1} OM (organic matter) were found. Cyanobacteria and algae were also of low abundance [4.2 cells mg^{− 1} DW (dry weight)]. Cyanobacteria comprised the major proportion of the autophotothrophic assemblages of subglacial soils. Deglaciated soils were similar to subglacial sediment in physico-chemical properties and microbial structure and numbers, unlike soil from vegetated sites or cryoconite sediment. In subglacial and deglaciated soil, relatively low diversity of microorganisms and low substrate availability was detected by PLFA analyses. Good accordance in microbial community structure assessments between epifluorescence microscopy and PLFA analyses was found. Our results suggest that the subglacial microbial populations can be divided into two groups: autochthonous microorganims (chemoheterotrophic bacteria) and allochthonous that retain the ability to proliferate and give rise to active population when conditions become favorable. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Estimating Virus Production Rates in Aquatic Systems

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative estimates of this process is critical as we can then develop better models of microbial community structure and function as well as advance our understanding of how viruses work to alter aquatic biogeochemical cycles. The virus reduction technique allows researchers to estimate the rate at which virus particles are released from the endemic microbial community. In brief, the abundance of free (extracellular) viruses is reduced in a sample while the microbial community is maintained at near ambient concentration. The microbial community is then incubated in the absence of free viruses and the rate at which viruses reoccur in the sample (through the lysis of already infected members of the community) can be quantified by epifluorescence microscopy or, in the case of specific viruses, quantitative PCR. These rates can then be used to estimate the rate of microbial mortality due to virus-mediated cell lysis.     

Use of the Malthus conductance growth analyser to determine numbers of thermophilic streptococci on stainless steel

The use of the Malthus conductance growth analyser for the detection of Streptococcus bovis attached to stainless steel surfaces was evaluated. A comparison between the results from acridine orange epifluorescence direct counts, swab recovery viable count and conductance estimates of attached cell concentrations, based on calibrations for planktonic cells, showed that the conductance results were up to 2 log₁₀ greater than the epifluorescence results and the swab counts. The growth rates of planktonic and attached cells were similar over 16 h using the Malthus technique. This suggests that the Malthus technique detects more attached cells of Strep. bovis than epifluorescence microscopy or swab recovery.